Determination of allelic diversity of candidate genes responsible for resistance to iron chlorosis by SSCP markers in some citrus rootstocks

We performed SSCP (Single Stranded Conformation Polimorphism) analysis in order to discover allelic diversity of candidate genes responsible for iron chlorosis in citrus rootstocks. Three Iron chlorosis candidate genes were selected from existing Citrus ESTs databases and Arabidopsis thaliana genome databases. Iron-Sulfur assembly protein, Vacuolar Metal Transporter and root iron transporter candidate genes were used. Eight citrus genotypes were selected to be representative cultivars of the basic taxon of Citrus to analyse the polymorphism. Two primers were used to amplify for each gene with these eight genotypes and directly sequencing of amplified DNA of candidate genes was done from PCR products to design new SSCP primers. We developed new primers for further diversity and routine genotyping by SSCP method from the results of direct sequencing on the basic taxa. Two pair of primers were defined for each genes. SSCP-PCR analysis was performed with thirty-five citrus rootstocks and PCR product were was subjected to electrophoresis in a non-denaturing PAGE and gels were stained with silver nitrate. Neighbour joining method was used to construct dendrograms for cluster analyses. Trifoliata genotypes and their hybrids were clustered together and mandarins showed high diversity for both genes, for Fe-S gene and IRT1 gene. Dendograms could not be constructed, because the DNA profiles could not be separated in acyrlamide gel for NRAMP3 gene.Key Words: SSCP, Iron Chlorosis, Citrus, Allelic diversity